III. EVALUATION ON SENSITIVITY OF SELECTED BIOMARKERS, CHOLINESTERASE ACTIVITY AND
SIPHONING RATE OF Mytilus edulis TO TRICHLORFON
3.1. Abstract
World widely used organophosphorus pesticides have been recognized as anticholinesterase of target and non-target organisms. The current study
demonstrated that organophosphorus pesticide, trichlorfon caused effects on both behavioral and cellular responses or biomarkers from blue mussels Mytilus edulis.
The mussels were exposed to serial dilutions of the pesticides i.e., 0, 50, 100, 200, 500, and 1000
μgl for 96 h. The notable behavior of mussels namely siphoning rate was inhibited at 200
μgl concentration. However, the effects of the pesticide on the behavior no longer existed after the mussels were transferred to clean
media. At cellular level, the experiment revealed that the pesticide induced effects on cholinesterase ChE activity of different organs at different
concentrations. A significant inhibition of the ChE activity from gill occurred at the lowest concentration i.e., 50
μgl, indicating that gill was the most sensitive organ. The moderate sensitive organs were foot and mantle, which elucidated the
significant effects of 200 μgl. The most insensitive organs were hemolymph,
posterior adductor muscle PAM and digestive gland. The ChE activities from the three organs were inhibited by trichlorfon at the highest concentration, 1000
μgl. After incubating the trichlorfon exposed mussels for seven days in clean media, the ChE activity from different organs of mussels was not cured
completely. The ChE activity from hemolymph, gill, PAM and digestive gland recovered, while the persistence of inhibited ChE activity from foot and mantle
were observed. The product moment Pearson correlation indicated the relationships between the siphoning rate and the ChE activity from three relevant
organs which are mantle, gill and PAM under exposure of trichlorfon.
Considering that the siphoning activity is the product of the three relevant organs movements, Backward Multiple Regression was applied to know which organ
play a dominant role in the siphoning activity. The Backward Multiple Regression emphasized the Pearson procedure by indicating the dominant role of
mantle in the siphoning activity. The implication of the use of the selected biomarkers on relevant organs in field and laboratory studies was discussed.
Keywords: Selected biomarker, siphoning rate, cholinesterase activity, trichlorfon, blue mussel.
3.2. Introduction
The extensive use of organophosphorous OP pesticides in agricultural and other antrophogenic activities causes increase of discharged pesticides wastes
in environment. Proclivity of the OP pesticides application in agricultural activities due to the fact, that the pesticides are not relatively persistent in
environment and effective for controlling and eradicating the pest. The considerable capability of the OP to control the pest is because of deliberate
synthesis of the pesticides to disrupt a neurotransmitter enzyme, viz. acetylcholinesterase AChE of the target animals. In fact, the enzyme has an
ability to hydrolyse huge numbers of neurotransmitter compounds i.e. acetylcholine in a neuromuscular junction for very short time Chang and
Strichartz 2005. Consequently, in exposed animals the neurotocxic actions of the OP, which inhibit AChE activity lead to excessive accumulation of
acetylcholine in postsynaptic cleft and hyperpolarisation of the postsynaptic membranes, which ultimately hinders and blocks the transmission of nerve
impulse. Although the occurrence of OP pesticides in the environment is thought to
be unstable compared to organochlorine pesticides, the persistent effects of the pesticides in non-target organism and ecological system cannot be ruled out
Scholz and Hopkins 2006. Once OP pesticides enter the body of organism most of them are transformed in metabolites which in many cases are more toxic
compounds than the parent compounds or induced directly to the target enzymes or organs Belden and Lydy 2000. Consequently, the effects of most of the
pesticides on AChE activity are considered as an irreversible action since the time of re-synthesis of the enzyme are naturally longer than the duration of dissociation
of the OP-Complex Gaglani and Bocquene 2000. Deteriorate effects of the pesticides become more prominent when dealkylation or what it called ageing
occurs which involves cleavage of an alkyl group of the phosphoryl moiety and the formation of negative charge, which stabilizes it Ray 1998. De novo
synthesis of the enzyme is the only way to recover AChE activity in the synaptic cleft, which goes slower than dealkylation reaction.
In aquatic ecosystem, the mechanisms of the OP actions in aquatic organisms such as Mytilus sp in the enzymatic levels, particularly cholinesterase
ChE activity, provide a comfortable tool as a biomarker for detection of the pesticide impacts in that ecosystem. Since the works of Grigor’eva and co-
workers, 1968, on ChEs from cardiac muscle and hemolymph of Mytilus edulis Moralev and Rozengrat 2004 and Wachtendonk and Neef 1979 on ChEs from
hemolymph, the ChE activity from the mussels has been explored and employed as a biomarker to detect the effects of OP pesticides in laboratory and field study.
Different organs of the mussels have been used to evaluate the detrimental effects of the OP pesticides on the ChE activity, which showed that gill was more
frequently used compared to others organ or whole tissue Escartin and Porte 1997; Mc.Henery et al. 1997; Mora et al. 1999a; Mora et al. 1999b; Dizer et al.
2001; Kopecka et al. 2004. In addition, the correlation between the inhibitions of the ChE activity from gill and increasing usage of dichlorvos in marine culture
was proved Mc.Henery et al. 1997. However, compare to the tissue homogenate, hemolymph from M. edulis has higher AChE activity Galloway et
al . 2002, and can be used without sacrificing the animals. Hence, the authors
claimed that the AChE activity from the mussel hemolymp provides a rapid, relatively cost-effective, reliable, and, non-destructive tool to assess the exposure
of mussels to OP and carbamate pesticides. Despite the fact that this biomarker is able to elucidate the significant cellular impairment of OP impacts on the mussels,
the ecological relevance of this biomarker is difficult to be predicted Baird et al. 2007. Therefore, there is a requirement to combine the cellular biomarker such
ChE activity with a biomarker at higher level of biological organization hereby the ecological relevance of the pesticide effects on the sentinel organism can be
possibly estimated. Behavioral biomarkers are relevant biological determinants for estimating
the impact of OP pesticides, which may have a pertinent potency to reflect the pesticide effects ecologically Peakall 1992; Sibley et al. 2000. Since the
nervous system is an important biological system that almost underlying the physiological and mechanisms of behavior and the state of the effect mechanisms
of pesticides is neurotoxin, the potential of the contaminants to disturb animal behavior is high Grue et al. 2002. Eventually, disturbance of behavior
performances of key species particularly due to the pesticide impact causes reduction of Darwinian fitness of the organisms, which would be manifested
potentially to the loss of ecological functions through decreasing population viability Baird et al. 2007.
Siphoning rate of bivalve is one of behavioral biomarker that has a close relationship to the mussel growth through energy acquisition Lagadic et al.
1994. It has been used to recognize the effects of the OP pesticide in M. edulis
McHenery et al. 1997; Donkin et al. 1997. Although Mc.Henery et al. 1997 could not detect the effect of the OP pesticide to which mussels filtered bacteria,
Donkin et al. 1997 detected clearly the effect of dichlorvos to the siphoning rate of mussels using algae. Nevertheless, the correlations between the ChE activity
from gill and the siphoning rate of M. edulis could not be observed Donkin et al. 1997. In the Asiatic clam, Corbicula fluminea, the interaction between the ChE
activity from the anterior and posterior adductor muscles and the siphoning rate were complicated by the closure of the valves when the clams were exposed to
higher concentrations of chlorpyrifos Cooper and Bidwell 2006. This experiment demonstrated that at lower concentrations the inhibition of the ChE
activity was followed by the insignificant inhibition of the siphoning rate. In contratst, at higher concentrations the significant inhibition of the siphoning rate
was associated with the intact ChE activity. Those studies implied that the ChE activity from gill and the adductor
muscles might not reflect directly to the siphoning rate and therefore the role of ChEs from gill and the adductor muscles on the siphoning rate regulation was
probably rather insignificant. On that account, the use of other organs of mussels that were possibly involved in the siphoning rate regulation such as mantle to
predict the pesticide effects in relation to ChE activity is indispensable. Furthermore, the serial dilutions of contaminant were used in the experiment
should be arranged properly to avoid behavior disturbances of mussels on estimation of the contaminant effects particularly in the cellular levels.
Having taking into account what have been explained above, the recent study was addressed mainly to evaluate:
1. The effects of the OP pesticide, viz. trichlorfon on the ChE activities on different organs and the siphoning rate of M. edulis.
2. The recovery from the pesticide impact on the ChE activities and the siphoning rate after the animals were transferred to clean media.
3. The correlation between the ChE activities in selected organs and the siphoning rate of M. edulis after exposure to trichlorfon.
3.3. Material and Methods Chemicals
